Species of Didymellaceae have a cosmopolitan distribution and are geographically widespread, occurring in diverse ecosystems. The family includes several important plant pathogenic fungi associated with fruit, leaf, stem and root diseases on a wide variety of hosts, as well as endophytic, saprobic and clinically relevant species. The Didymellaceae was recently revised based on morphological and phylogenetic analyses of ex-type strains subjected to DNA sequencing of partial gene data of the LSU, ITS, rpb2 and tub2 loci. Several poly- and paraphyletic genera, including Ascochyta, Didymella and Phoma were redefined, along with the introduction of new genera. In the present study, a global collection of 1124 Didymellaceae strains from 92 countries, 121 plant families and 55 other substrates, including air, coral, human tissues, house dust, fungi, insects, soil, and water were examined via multi-locus phylogenetic analyses and detailed morphological comparisons, representing the broadest sampling of Didymellaceae to date. Among these, 97 isolates representing seven new genera, 40 new species and 21 new combinations were newly introduced in Didymellaceae. In addition, six epitypes and six neotypes were designated to stabilise the taxonomy and use of older names. A robust, multi-locus reference phylogenetic tree of Didymellaceae was generated. In addition, rpb2 was revealed as the most effective locus for the identification of Didymellaceae at species level, and is proposed as a secondary DNA marker for the family. Key words: Multi-locus phylogeny, New taxa, Phoma, rpb2, Taxonomy. Taxonomic novelties: New genera: Dimorphoma L.W. Hou, L. Cai & Crous, Ectodidymella L.W. Hou, L. Cai & Crous, Longididymella L.W. Hou, L. Cai & Crous, Macroascochyta L.W. Hou, L. Cai & Crous, Paramicrosphaeropsis L.W. Hou, L. Cai & Crous, Pseudopeyronellaea L.W. Hou, L. Cai & Crous, Sclerotiophoma L.W. Hou, L. Cai & Crous. New species: Allophoma alba L.W. Hou, Pfenning, L. Cai & Crous, Al. anatii L.W. Hou & O. Yarden, Ascochyta pilosella L.W. Hou, L. Cai & Crous, Calophoma parvula L.W. Hou, L. Cai & Crous, Chaetasbolisia argentina L.W. Hou, L. Cai & Crous, Didymella aloeicola L.W. Hou, L. Cai & Crous, Did. guttulata L.W. Hou, L. Cai & Crous, Did. indica L.W. Hou, L. Cai & Crous, Did. mitis L.W. Hou, L. Cai & Crous, Did. prolaticolla L.W. Hou, L. Cai & Crous, Did. subglobispora L.W. Hou, L. Cai & Crous, Did. subrosea L.W. Hou, L. Cai & Crous, Did. variabilis L.W. Hou, L. Cai & Crous, Epicoccum brahmansense L.W. Hou, L. Cai & Crous, Ep. dickmanii L.W. Hou & O. Yarden, Ep. longiostiolatum L.W. Hou, L. Cai & Crous, Ep. multiceps L.W. Hou, L. Cai & Crous, Ep. polychromum L.W. Hou, L. Cai & Crous, Ep. variabile L.W. Hou, L. Cai & Crous, Leptosphaerulina obtusispora L.W. Hou, L. Cai & Crous, Le. sisyrinchiicola L.W. Hou, L. Cai & Crous, Macroascochyta grandis L.W. Hou, L. Cai & Crous, Macroventuria angustispora L.W. Hou, L. Cai & Crous, Ma. terrestris L.W. Hou, L. Cai & Crous, Microsphaeropsis fusca L.W. Hou, L. Cai & Crous, Mi. taxicola L.W. Hou, L. Cai & Crous, Mi. viridis L.W. Hou, L. Cai & Crous, Neoascochyta fusiformis L.W. Hou, L. Cai & Crous, Neoa. humicola L.W. Hou, L. Cai & Crous, Neoa. longispora L.W. Hou, L. Cai & Crous, Neoa. mortariensis L.W. Hou, L. Cai & Crous, Neodidymelliopsis tiliae L.W. Hou, L. Cai & Crous, Nothophoma infuscata L.W. Hou, L. Cai & Crous, Paramicrosphaeropsis ellipsoidea L.W. Hou, L. Cai & Crous, Phomatodes pilosa L.W. Hou, L. Cai & Crous, Remotididymella brunnea L.W. Hou, L. Cai & Crous, R. humicola L.W. Hou, L. Cai & Crous, Xenodidymella glycyrrhizicola L.W. Hou, L. Cai & Crous, Stagonosporopsis cucumeris L.W. Hou, L. Cai & Crous, St. sambucella L.W. Hou, L. Cai & Crous. New combinations: Ascochyta astragalina (Rehm ex Sacc.) L.W. Hou, L. Cai & Crous, As. koolunga (J.A. Davidson et al.) L.W. Hou, L. Cai & Crous, Calophoma vincetoxici (De Not.) L.W. Hou, L. Cai & Crous, Chaetasbolisia eupatorii (Died.) L.W. Hou, L. Cai & Crous, Didymella combreti (Crous) L.W. Hou, L. Cai & Crous, Did. prosopidis (Crous & A.R. Wood) L.W. Hou, L. Cai & Crous, Dimorphoma saxea (Aveskamp et al.) L.W. Hou, L. Cai & Crous, Ectodidymella nigrificans (P. Karst.) L.W. Hou, L. Cai & Crous, Ectophoma insulana (Mont.) L.W. Hou, L. Cai & Crous, Epicoccum tobaicum (Szilv.) L.W. Hou, L. Cai & Crous, Longididymella clematidis (Woudenb. et al.) L.W. Hou, L. Cai & Crous, Lo. vitalbae (Briard & Har.) L.W. Hou, L. Cai & Crous, Nothophoma acaciae (Crous) L.W. Hou, L. Cai & Crous, No. eucalyptigena (Crous) L.W. Hou, L. Cai & Crous, No. prosopidis (Crous & A.R. Wood) L.W. Hou, L. Cai & Crous, Pseudopeyronellaea eucalypti (Crous & M.J. Wingf.) L.W. Hou, L. Cai & Crous, Remotididymella capsici (Bond.-Mont.) L.W. Hou, L. Cai & Crous, Sclerotiophoma versabilis (Boerema et al.) L.W. Hou, L. Cai & Crous, Stagonosporopsis nemophilae (Neerg). L.W. Hou, L. Cai & Crous, Vacuiphoma ferulae (Pat.) L.W. Hou, L. Cai & Crous, Va. laurina (Thum.) L.W. Hou, L. Cai & Crous. New name: Heterophoma verbasci-densiflori L.W. Hou, L. Cai & Crous, Nothophoma nullicana L.W. Hou, L. Cai & Crous. Epitypes (basionyms): Amphisphaeria vincetoxici De Not., Phoma eupatorii Died., Phyllosticta acetosellae A.L. Sm. & Ramsb., Phyllosticta verbascicola Ellis & Kellerm., Phyllosticta arachidis-hypogaeae V.G. Rao, Pleosphaerulina briosiana Pollacci. Neotypes (basionyms): Ascochyta ferulae Pat., Ascochyta nobilis Kabat & Bubak, Epicoccum oryzae S. Ito & Iwadare, Phoma laurina Thum., Phoma nemophilae Neerg., Phyllosticta insulana Mont. Lectotypes (basionyms): Amphisphaeria vincetoxici De Not., Phoma eupatorii Died. Resurrected names: Epicoccum mezzettii Goid., Ep. oryzae Ito & Iwadare, Ep. purpurascens Ehrenb., Toruloidea tobaica Szilv.